Live-Cell Quantification of Mitochondrial Functional Parameters

Mitochondria are semi-autonomous organelles, which are central to cellular energy production and signal transduction. Given the tight integration between mitochondrial and cellular physiology, experimental strategies are required to study mitochondrial (dys)function in living cells. For this purpose one can use various chemical and protein-based fluorescent reporter molecules (probes), which are introduced into the cell using specific incubation protocols or transfection techniques. These probes include reporters to monitor mitochondrial membrane potential (Δψ ), cytosolic and mitochondrial free calcium concentration (Ca²⁺ ), reactive oxygen species (ROS), cytosolic and mitochondrial pH, glucose and ATP. However, proper interpretation and quantification of the above readouts is not trivial. Here, we present our protocol for automated temporal analysis of mitochondrial position in living cells and explain how it can be used for computer-assisted quantification of mitochondrial morphology and Δψ . We further discuss how this approach can be applied for simultaneous quantification of multiple mitochondrial and cellular parameters.